Mutational disruption of plasma membrane trafficking of Saccharomyces cerevisiae Yor1p, a homologue of mammalian multidrug resistance protein. (57/10751)

The ATP binding cassette (ABC) transporter protein Yor1p was identified on the basis of its ability to elevate oligomycin resistance when it was overproduced from a high-copy-number plasmid. Analysis of the predicted amino acid sequence of Yor1p indicated that this protein was a new member of a subfamily of ABC transporter proteins defined by the multidrug resistance protein (MRP). In this work, Yor1p is demonstrated to localize to the Saccharomyces cerevisiae plasma membrane by both indirect immunofluorescence and biochemical fractionation studies. Several mutations were generated in the amino-terminal nucleotide binding domain (NBD1) of Yor1p to test if the high degree of sequence conservation in this region of the protein was important for function. Deletion of a phenylalanine residue at Yor1p position 670 led to a mutant protein that appeared to be retained in the endoplasmic reticulum (ER) and that was unstable. As shown by others, deletion of the analogous residue from a second mammalian MRP family member, the cystic fibrosis transmembrane conductance regulator (CFTR), also led to retention of this normally plasma membrane-localized protein in the ER. Changes in the spacing between or the sequences flanking functional motifs of Yor1p NBD1 led to defective trafficking or decreased activity of the mutant proteins. Analyses of the degradation of wild-type and DeltaF670 Yor1p indicated that the half-life of DeltaF670 Yor1p was dramatically shortened. While the vacuole was the primary site for turnover of wild-type Yor1p, degradation of DeltaF670 Yor1p was found to be more complex with both proteasomal and vacuolar contributions.  (+info)

Functional analysis of mutations conferring lamivudine resistance on hepatitis B virus. (58/10751)

Two patterns of mutation are commonly observed in the polymerase gene of lamivudine [(-)2'-deoxy-3'-thiacytidine]-resistant hepatitis B virus (HBV). The M539I substitution in the conserved YMDD motif occurs independently of other changes, whereas the M539V substitution is associated with an additional upstream change (L515M). These mutations were introduced into a common background and their effects on HBV DNA replication and lamivudine resistance studied. The L515M and M539V mutations provided only partial resistance while the M539I mutation conferred a high degree of lamivudine resistance. The combination of the L515M and M539V mutations gave an intermediate level of replication competence, compared with either mutation alone, and increased resistance to lamivudine. This probably accounts for these two mutations always being observed together. The M539I mutation reduced replication competence.  (+info)

Altered expression profile of the surface glycopeptidolipids in drug-resistant clinical isolates of Mycobacterium avium complex. (59/10751)

Members of the Mycobacterium avium complex are the most frequently encountered opportunistic bacterial pathogens among patients in the advanced stage of AIDS. Two clinical isolates of the same strain, numbers 397 and 417, were obtained from an AIDS patient with disseminated M. avium complex infection before and after treatment with a regimen of clarithromycin and ethambutol. To identify the biochemical consequence of drug treatment, the expression and chemical composition of their major cell wall constituents, the arabinogalactan, lipoarabinomannan, and the surface glycopeptidolipids (GPL), were critically examined. Through thin layer chromatography, mass spectrometry, and chemical analysis, it was found that the GPL expression profiles differ significantly in that several apolar GPLs were overexpressed in the clinically resistant 417 isolate at the expense of the serotype 1 polar GPL, which was the single predominant band in the ethambutol-susceptible 397 isolate. Thus, instead of additional rhamnosylation on the 6-deoxytalose (6-dTal) appendage to give the serotype 1-specific disaccharide hapten, the accumulation of this nonextended apolar GPL probably provided more precursor substrate available for further nonsaccharide substitutions including a higher degree of O-methylation to give 3-O-Me-6-dTal and the unusual 4-O-sulfation on 6-dTal. Further data showed that this alteration effectively neutralized ethambutol, which is known to inhibit arabinan synthesis. Thus, in contrast with derived Emb-resistant mutants of Mycobacterium smegmatis or Mycobacterium tuberculosis, which are devoid of a surface GPL layer, the lipoarabinomannan from resistant 417 isolate grown in the presence of this drug was not apparently truncated.  (+info)

Differential sensitivity of distinct Chlamydia trachomatis isolates to IFN-gamma-mediated inhibition. (60/10751)

Resistance to the mouse pneumonitis (MoPn) strain of Chlamydia trachomatis has been mapped to MHC class II-restricted, IL-12-dependent CD4+ T cells that secrete a type 1 profile of proinflammatory cytokines, which includes IFN-gamma and TNF-alpha. The relative contribution of IFN-gamma is controversial, however, due to variation in results presented by different laboratories. To determine whether C. trachomatis strain differences contributed to this apparent conflict, the relative resistance of IFN-gamma-deficient mice to murine and human strains of C. trachomatis was compared. All human serovars were much more sensitive to the direct inhibitory actions of IFN-gamma than the MoPn strain. Furthermore, genital clearance of human serovar D in the C57BL/6 mouse was mediated by class II-independent mechanisms that probably involved local production of IFN-gamma by cells of the innate immune system. TNF-alpha also contributed indirectly to host resistance against all strains tested. The differential susceptibility of distinct C. trachomatis strains to effector cytokines such as IFN-gamma could not have been predicted by interstrain biologic variation or by the profile of cytokines stimulated during infection. These findings indicate that strain variation should be considered in situations where related isolates of a given parasite produce conflicting data in models of infection and immunity. They also suggest that stimulation of mucosal IFN-gamma activity is a relevant goal for a human chlamydial vaccine.  (+info)

Role of acid pH and deficient efflux of pyrazinoic acid in unique susceptibility of Mycobacterium tuberculosis to pyrazinamide. (61/10751)

Pyrazinamide (PZA) is an important antituberculosis drug. Unlike most antibacterial agents, PZA, despite its remarkable in vivo activity, has no activity against Mycobacterium tuberculosis in vitro except at an acidic pH. M. tuberculosis is uniquely susceptible to PZA, but other mycobacteria as well as nonmycobacteria are intrinsically resistant. The role of acidic pH in PZA action and the basis for the unique PZA susceptibility of M. tuberculosis are unknown. We found that in M. tuberculosis, acidic pH enhanced the intracellular accumulation of pyrazinoic acid (POA), the active derivative of PZA, after conversion of PZA by pyrazinamidase. In contrast, at neutral or alkaline pH, POA was mainly found outside M. tuberculosis cells. PZA-resistant M. tuberculosis complex organisms did not convert PZA into POA. Unlike M. tuberculosis, intrinsically PZA-resistant M. smegmatis converted PZA into POA, but it did not accumulate POA even at an acidic pH, due to a very active POA efflux mechanism. We propose that a deficient POA efflux mechanism underlies the unique susceptibility of M. tuberculosis to PZA and that the natural PZA resistance of M. smegmatis is due to a highly active efflux pump. These findings may have implications with regard to the design of new antimycobacterial drugs.  (+info)

Mutations that confer resistance to 2-deoxyglucose reduce the specific activity of hexokinase from Myxococcus xanthus. (62/10751)

The glucose analog 2-deoxyglucose (2dGlc) inhibits the growth and multicellular development of Myxococcus xanthus. Mutants of M. xanthus resistant to 2dGlc, designated hex mutants, arise at a low spontaneous frequency. Expression of the Escherichia coli glk (glucokinase) gene in M. xanthus hex mutants restores 2dGlc sensitivity, suggesting that these mutants arise upon the loss of a soluble hexokinase function that phosphorylates 2dGlc to form the toxic intermediate, 2-deoxyglucose-6-phosphate. Enzyme assays of M. xanthus extracts reveal a soluble hexokinase (ATP:D-hexose-6-phosphotransferase; EC 2.7.1.1) activity but no phosphotransferase system activities. The hex mutants have lower levels of hexokinase activities than the wild type, and the levels of hexokinase activity exhibited by the hex mutants are inversely correlated with the ability of 2dGlc to inhibit their growth and sporulation. Both 2dGlc and N-acetylglucosamine act as inhibitors of glucose turnover by the M. xanthus hexokinase in vitro, consistent with the finding that glucose and N-acetylglucosamine can antagonize the toxic effects of 2dGlc in vivo.  (+info)

Vibrio cholerae O1 El Tor: identification of a gene cluster required for the rugose colony type, exopolysaccharide production, chlorine resistance, and biofilm formation. (63/10751)

The rugose colony variant of Vibrio cholerae O1, biotype El Tor, is shown to produce an exopolysaccharide, EPSETr, that confers chlorine resistance and biofilm-forming capacity. EPSETr production requires a chromosomal locus, vps, that contains sequences homologous to carbohydrate biosynthesis genes of other bacterial species. Mutations within this locus yield chlorine-sensitive, smooth colony variants that are biofilm deficient. The biofilm-forming properties of EPSETr may enable the survival of V. cholerae O1 within environmental aquatic habitats between outbreaks of human disease.  (+info)

Surveillance of resistant pathogens and rational use of antibiotics: general remarks. (64/10751)

Surveillance of resistant pathogens should lead to improved treatment of patients and to a rational use of antibiotics. The process for decision making between microbiology, general practice and health policy is still to be documented with careful studies.  (+info)